大头蛙和脆皮大头蛙线粒体3个基因的测定及两栖类亲缘关系研究

现存的两栖类系统发生关系一直存在争议,特别是3个目间的亲缘关系。本文设计了5对引物,扩增和测定了大头蛙和脆皮大头蛙线粒体12S和16S rRNA基因和Cytb基因的全序列。在对所测序列进行分析的同时,基于3个基因全序列的相加数据,运用MEGA 3.1和PHYLIP 3.64软件中的NJ法、MP法和ML法,对两爬类17个物种,以鱼类非洲肺鱼为外群,重建出3个树形完全一致的分子系统树。研究结果显示:现存两栖类中无尾目和有尾目为姐妹群关系,并推断有尾目内小鲵科和隐鳃鲵科亲缘关系较近。此外,在研究两栖类系统发生关系方面,作者分析前人研究中产生两种不同观点的可能原因,同时总结了在此类研究中产生偏差的几种影响因素。

Sequence of three mitochondrial genes of L. kuhlii and L. fragilis, and the phylogenetic relationships among frogs, salamanders and caecilians

At the present time,the phylogenetic relationships among three living amphibian orders,3 families of Caudate are still debated.To test the phylogenetic relationships among these modern amphibians(frogs,salamanders and caecilians),based on the PCR fragments of 5 primers we designed,the complete sequences of mitochondrial 12S rRNA,16S rRNA and Cytb genes were determined for L.kuhlii and L.fragilis.In L.kuhlii the 12S and 16S rRNAs were 928 and 1586 nt long,respectively.The corresponding genes were 924 and 1579 bp long in L. fragilis.The overall A T content of two rRNAs of L.kuhlii was 57.74%,and that of L.fragilis was 56.63%.The nucleotide sequence of L.kuhlii 12S and 16S rRNAs had 84.15% similarity to the homologous sequence of L.fragilis.The Cytb genes began with an ATG start codon and used complete TAA as a stop codon in both sequences of two frogs.The sequences of this gene showed a 23.01% divergence at the nucleotide level and 9.40% divergence at the amino acid level between two frogs.In addition,located upstream and downstream of 12S rRNA genes 4 complete tRNA genes were obtained for the two frogs(two tRNAPhe and two tRNAVal respectively),and all could fold in a canonical cloverleaf secondary structure.Two sequences of L.kuhlii and L.fragilis including several genes had been deposited in the GenBank database under the accession number AY899241 and AY899242,respectively.When African lungfish(Protopterus dolloi NC-001708) was selected as an outgroup,and compared with the same mtDNA fragments of 13 other amphibians(five frogs: Bufo melanostictus NC-005794,Hyla Chinensis AY458593,Polypedates megacephalus AY458598,Rana nigromaculate AB043889 and Fejervarya.limnocharis NC-005055; 5 salamanders: Mertensiella luschani AF154053,Ambystoma mexicanum AY659991,Andrias davidianus AJ492192,Andrias japonicus AB208679 and Ranodon sibricus AJ419960;3 caecilians: Typhlonectes natans AF154051,Ichthyophis bannanicus AY458594 and Uraeotyphlus cf.oxyurus NC-006305) and 2 species of reptiles(Alligator mississippiensis Y13113 and Eumeces egregius AB016606) recorded in the GenBank,we reconstructed phylogenetic trees using MEGA version 3.1 and PHYLIP version 3.64,based on a data set combining two rRNA genes and Cytb genes.The phylogenetic analyses results showed that NJ,MP and ML trees all strongly supported a sister group relationship for frogs and salamanders,and favored a Cryptobranchidae Hynobiidae clade with high bootstap support values.The result congruent with the traditional hypothesis(the Batrachia hypothesis),and it was also supported by previous molecular studies based on the data mitochondrial genes(i.e.,based on a data set combining 12 proteins and 2 rRNA,and every major amphibian group contained at least two species).In addition,the reason for our result inconsistent with previous works and the shortcomings of phylogenetic analyses based on the different mitochondrial genes,and selected the different species of the major amphibian groups and outgroups were also discussed.So,future researches on the evolutionary and phylogenetic relationships of amphibian will no doubt need more evidence of morphological and fossil data,and a larger molecular data set,including more genes and key species.